Diagnosis and treatment of HIV viral infection using magnetic metal transferrin particles

ABSTRACT

This invention provides methods of treatment and/or diagnosis and/or siting of viruses including the AIDS virus and others as well as the cells which they infect. The method comprises introducing near, into or onto the virus or the cell which the virus infects, or both, minute particles. These particles possess ferromagnetic, paramagnetic or diamagnetic properties. After being localized near, in or on the virus or the viral-infected cell, the particles are inductively heated by application of an alternating electromagnetic field. The inductive heating is continued for a period of time sufficient to bring about a temperature rise to a minimum necessary to kill the virus or cell or to desirably alter the behavior of the virus or infected cell. Prior to, during or after treatment, these particles can be used diagnostically to locate and/or map the virus in the living tissue.

This is a continuation of application Ser. No. 08/038,037 filed Mar. 29,1993, now abandoned, which in turn is a continuation of U.S. Ser. No.07/792,474, filed Nov. 15, 1991 (now abandoned), which is acontinuation-in-part of U.S. Ser. No. 07/285,979, filed Dec. 19, 1988(now abandoned).

BACKGROUND OF THE INVENTION

Presently, one of the most serious illnesses facing the world is theviral infection, Acquired Immunodeficiency Syndrome (AIDS). Thisinfection is caused by a virus, designated HIV, or one of its variants.These contain RNA which is coated with a protein shell. The virusaffects the T-cells (lymphocytes) of the body as well as otherimmune-oriented cells (i.e., glial cells in the brain). This effectresults in a serious reduction of the body's immunological ability tofight disease and the various bacterial, viral and fungal entities whichconstantly are in the environment. The fact that a virus becomes a partof cells themselves makes it difficult to affect them adversely withoutharm to the host. Current methods of treatment involve the use ofchemotherapeutic agents. AZT is one choice, but the side-effects arevery significant.

The AIDS virus causes problems in an infected individual through theproduction of alterations in the cell function. These alterations areproduced through the interaction of the virus and subcellularcomponents, nuclear protein being one example.

A method of treatment of the virus and viral-infected cells is clearlydesirable. To be completely successful, the treatment should kill theinvading organism while causing substantially no harm to host tissue.Also, a treatment should produce little or no chance for the organismbeing treated to become tolerant or resistant to the treatment method.

One method applied to organisms in general, including viruses, is tofocus upon a particular aspect of the infectious organism's metabolismwhich differs from that of the host cells. Rather than interfering withcellular pathways as antibiotics do, this method exploits the organism'sroutine use of that pathway in such a way that it may be turned againstthe organism, thus killing it. See U.S. Pat. No. 4,590,922 of R. T.Gordon. However, it is not disclosed that this method is applicable toHIV and related viruses.

SUMMARY OF THE INVENTION

This invention relates, inter alia, to a method of treating the AIDSvirus, HIV and related organisms (e.g., HTLV and others), or cellsinfected therewith, comprising providing said organisms and/or cellswith minute, inductively heatable, intracellularly localizable particlesof a size less than 1 micron and inductively heating said particles bythe application of an alternating electromagnetic field (or oscillatingor pulsed) for a period of time sufficient to effect a rise inintracellular temperature to a minimum necessary to kill the infectiousorganisms. Independent investigators have determined that it requires atemperature of approximately 60° C. for a period of 30 minutes toincapacitate or kill the HIV virus. (Spire et al., The Lancet, Jan. 26,1985, 188; Po Collinson et al., Ann. Clin. Biochem. 1986, 23:102; andHoussein et al., Clinical Chemistry, Vol. 31, No. 12, 1985). Thisinvention readily achieves this selectivity without heating the entirebody to this intolerable temperature. In addition to raising HIVtemperature to a given level for a time sufficient to kill the virus,the temperature can be raised to a level for a time sufficient tocauterize it. Other temperature levels/times, e.g., sufficient toinactivate the virus can be used also.

The particles best suited for this treatment are pharmacologicallyacceptable ferromagnetic, paramagnetic, including superparamagnetic, ordiamagnetic particles, e.g., suspended e.g., colloidally, in a liquidvehicle. These possess magnetic properties uniquely suited for treatmentand diagnostic regimens. Many such particles are disclosed in R. T.Gordon's U.S. Pat. Nos. 4,106,488; 4,136,683; 4,303,636, 4,569,836,4,731,239, 4,735,796 and 4,590,922, all of which disclosures are fullyincorporated by reference herein.

These particles may be selected from ferromagnetic, paramagnetic ordiamagnetic inorganic elements and compounds as well as organiccompounds such as metal dextran complexes, metal-containing prostheticgroups, transport or storage proteins and the like. The invention mayutilize particles exogenously supplied to the infected cells or viralorganisms, or particles which are endogenous to the infected cells,e.g., in the form of one of the above-mentioned elements or compounds.The treatment is particularly useful for HIV virus or HIV virus-infectedcells.

The present invention provides a technique for achieving a preciseincrement of heat or energy rise within the virus itself or thevirus-infected cell. The internal temperature of the invading HIV viruscan be raised to the minimum necessary to destroy it. Alternatively, thevital-infected cells can be destroyed or altered so as to resume normalfunction. In accordance with the present invention, there are a numberof approaches which can achieve the end result of destroying the virusaffecting the vital-infected cells without causing damage to the hostcells.

In its preferred aspect as mentioned above, the invention introduces,near, on or into the virus or the viral-infected cells, minute particles(e.g., less than 1 micron in diameter, e.g., on the order of about 0.1,1, 10, 100, 1000 or 10,000 Angstroms, or smaller, the precise size notbeing critical) of a ferromagnetic, paramagnetic or diamagneticmaterial. By "near" herein is meant sufficiently close to effect themethod of this invention.

Minute particles possessing ferromagnetic, paramagnetic or diamagneticproperties have already been shown to be particularly useful in treatingcancer, as described by R. T. Gordon, e.g., in U.S. Pat. Nos. 4,106,488and 4,735,796 mentioned above. As exemplified therein, ferric hydroxideand gallium citrate can be used to form particles of a size of 1 micronor less and may be introduced into the cancer cells in the area to betreated. The cells of the chosen area may then be subjected to ahigh-frequency alternating electromagnetic field which inductively heatsthe intracellular particles, resulting in an increase in intracellulartemperature. Because the cancer cells accumulate the particles to agreater degree than normal cells, and because they also have a higherresting temperature than normal cells, the increase in temperature killsthe cancer cells and leaves the normal cells substantially unharmed.This technique has also been applied by Gordon to infectious diseasesincluding those caused by bacteria, fungi, protozoa and viruses. (U.S.Pat. No. 4,590,922)

The present invention involves the unexpected discovery that, withcertain modification where appropriate, the intracellular hyperthermiatechnique as disclosed by Gordon may be effectively utilized indestroying the HIV virus or altering the infected cells' behavior, e.g.,by exploiting the uniqueness and/or specificity of some of the viruses'metabolic pathways and/or magnetically affectable, optionallymetabolizable, products, to selectively concentrate magnetic particleswithin cells infected by the AIDS-causing organisms. The invention alsoutilizes the technique by selectively focusing the inductive heatingprocess upon magnetic particles found in the HIV viruses orvirus-infected cells but substantially not in host cells or therein to asignificantly lesser degree. In addition, these particles and methodsmay be used diagnostically, e.g., to locate viral-containing cells inthe appropriate living tissue.

One example of such a selective pathway involves the way in whichviruses require iron for the infected cells to survive and proliferate.(This discussion of the invention in relation to iron metabolism is inno way intended to limit this invention which applies generally and notonly to iron metabolism.) The necessity of uptake of iron byviral-infected cells is well known (Blumberg et al, Lancet Feb. 11,1984, pg. 347). Actually, in the AIDS patient, it has been known thatserum iron levels fall due to the utilization of Fe by the infectiousorganisms. Serum ferritin also increases in this condition. In addition,in HIV infections, the T-cell lymphocyte is prominently involved. Butthe resting T-cell lymphocyte has very few receptors for transferrin,the glycoprotein which binds Fe for transport. However, when the T-cellis infected with HIV, the number of transferrin receptors increasestremendously. This increase in the number of transferrin receptors onthe activated T-lymphocytes is very important as will be discussedbelow.

Another example involves the binding of terbium to the nucleic acid ofRNA virus, e.g., as has been shown by Morley et al, Biochem. Biophys.Res. Commun. 101:1123, 1981. The binding of platinum-complexed particlesto nucleic acids has also been shown. Moreover, the binding ofPt-Rhodamine complexes to nucleic acid has been demonstrated.Metalloporphyrins also bind to the coating material. All of thesepathways can be used in conjunction with this invention, e.g., byincluding appropriate corresponding chemical entities in the particlesof this invention whereby these pathways are entered by the particlesresulting in localization of them in, near or on the HIV virus or cellsinfected therewith. Moreover, when the HIV virus or infected cellscontain ferromagnetic and/or paramagnetic elements within them, theseendogenous moieties can also be used to produce the effect of thisinvention.

Particularly useful particles for this invention include, as discussedabove, both inorganic elements and compounds as well as metal-containingorganic compounds. Inorganic elements and compounds particularlywell-suited, owing to their favorable magnetic parameters, compriseelements such as dysprosium, erbium, europium, gallium, holmium,samarium, terbium, thulium, ytterbium or yttrium and compounds thereof,such as dysprosium sulfate, erbium sulfate, europium oxide, europiumsulfate, holmium oxide, samarium sulfate, terbium oxide, terbiumsulfate, thulium oxide, ytterbium sulfide, yttrium oxide, yttriumsulfate, yttrium ferrioxide (Y₃ Fe₅ O₁₂) and yttrium aluminum oxide (Y₃Al₅ O₁₂).

Metal-containing organic molecules useful for the application discussedabove comprise particles of iron-dextrans such as FeOOH-dextrancomplexes and other dextran complexes and other dextran metal complexeswherein the metal is selected from the group comprising cobalt, zinc,chromium, nickel, platinum, manganese and rare earth metals such asdysprosium, erbium, europium, gallium, holmium, samarium, terbium,thulium, ytterbium and yttrium, ferric ammonium citrate, enterochelin,hydroxamates, phenolates, ferrichromes, ferritin (e.g., referring to theabove, to bind a glycoprotein envelope of a virus), ferric mycobactins,and iron-sulfur proteins such as ferredoxin and rubredoxin.

Particularly appropriate metal-containing organic structures for usewith the present invention are the porphyrins such as etioporphyrins,mesoporphyrins, uroporphyrins, coproporphyrins, protoporphyrins, anddicarboxylic acid containing porphyrins and substituted porphyrins suchas tetraphenylporphyrin sulfonate (TTPS). Especially advantageousprotoporphyrins comprise hematoporphyrins, chlorophylis and cytochromes.In addition to the naturally occurring protoporphyrins which possessiron- or magnesium-containing moieties, mixed metal hybrid porphyrinsmay also be prepared. For example, by substituting an alternative metalfor the iron in hematoporphyrin, the advantages of the porphyrin moiety(e.g., in terms of specificity of localization) is retained while theunique magnetic properties of the new metal enhance the sensitivity ofthe substituted molecule. Suitable metals for purposes of substitutioncomprise cobalt, manganese, zinc, chromium, nickel, platinum and rareearth series of metals, dysprosium, erbium, europium, gallium, holmium,samarium, terbium, thulium, ytterbium and yttrium. Suitable porphyrinacceptors comprise any dicarboxylic acid containing porphyrin, such asprotoporphyrins (e.g., hematoporphyrins) and the like.

Especially appropriate particles are the Fe₃ O₄ -dextran-transferrincompounds. Included in this group are magnetic particle transferrincompounds where the magnetic particle is ferromagnetic, paramagnetic ordiamagnetic. The magnetic particle transferrin may be bound to a dextranto enhance its uptake. Additional complexes found to be useful includeplatinum-rhodamine complexes, terbium complexes and heavy metalcomplexes, e.g., lanthanide complexes.

The principle upon which the present invention is based is grounded inthe discovery that the HIV virus or a host cell infected therewith willtransport, metabolize and/or sequester many elements or compounds inquite a different manner from that of the cells of the more advancedhost organisms it usually infects. Note that HTLV-I infections result inderegulation of surface expression of the transferrin receptor. (Vidal,J. Immun., 141, 984 (1988).) See also Gastl et al., Blut, 56:193 (1988).In one aspect of this invention, this specificity is thus used toselectively concentrate the above-mentioned particles within the virusor infected cells, with little or no uptake of said particles by normalhost cells.

Thus, as mentioned, the particles introduced into the virus orviral-infected cells will generally be infectious organism-specific,i.e., an element or compound peculiar to the metabolism of theAIDS-causing organism being treated. Compounds which are particularlyuseful in this regard are any of the above-mentioned metal-chelatingtransport substances specific to the viruses and/or viral-infectedcells. Also noteworthy in this process are such metal-containing organicstructures as the porphyrins, including hematoporphyrins, cytochromesand chlorophylis. In addition to the naturally occurring porphyrins,mixed metal hybrid porphyrins may be prepared, substituting manganese,zinc, cobalt, chromium, nickel, platinum and rare earth series of metalssuch as dysprosium, erbium, europium, gallium, holmium, samarium,terbium, thulium ytterbium and yttrium. See the foregoing.

The minute particles described can be administered to the patiententerally, e.g., orally, parenterally, e.g., intravenously,intramuscularly, intraperitoneally, subcutaneously, topically, insuppository form, etc., depending upon the nature and location of theinfection. Dosage and frequency of administration may also varydepending upon the nature of the HIV virus or viral-infected cells, ascan be routinely determined.

The next stage of the present invention, after particle localization, isdifferential killing of the virus and/or such killing, incapacitating,cauterizing or altering of the viral-infected cells by causing inductiveheating using a high frequency alternating electromagnetic fieldbringing about a precisely controllable rise in temperature or elevationin level of energy. Equivalently, pulsed electromagnetic fields andradiation (e.g., from a laser) and variants and combinations can also beemployed. The principle of inductive heating through the use ofhysteresis is a well known principle. Similarly, the monitoring of thetemperatures of the living cells is a presently available technique wellknown to medical science. See the Gordon patents.

Inductive heating of the minute particles is achieved through use of anelectric oscillator operating in the high frequency range which heatsthe particles by subjecting them to an intense high-frequency fieldwithin a large but otherwise conventional helical coil, field energybeing converted to heat through hysteresis losses and the resistivedissipation of eddy currents. The helical induction coil is ofsufficient internal diameter to permit the patient to pass within and ofsuch length to encompass the length of the patient. Preferably, theinternal diameter is greater than 3-6 feet, since diameters of inductivecoils greater than 6 feet have a preferential effect on the overallprocess by providing a more uniform flux gradient to the patient. Seethe Gordon patents. However, smaller diameters, e.g., about 1 meter orso are also applicable. Moreover, the length could be approximately 20centimeters with provision for scanning the body. Coils of other shapesstrategically placed could also be utilized in achieving the coupling ofthe electromagnetic energy to the particles.

The frequency of the electromagnetic alternating high-frequency fieldwill range from 1 kilohertz to 100 megahertz, preferably about 400 kHz,and the power input of the oscillator-generator will range from 1kilowatt to 30 kilowatts per kg of patient body weight. 1 kilowatt ofpower per 1.0 kilogram of body weight is particularly useful. In thispower and frequency range, the coils are selected to produce from 1 to5×10⁴ oersteds, preferably from 10 to 10,000 oersteds. Times aregenerally in the range of several seconds to several hours. The higherthe intensity, the shorter the time period necessary to kill the HIVvirus. Preferably, a high intensity, short time period will be used tocause a significant rise in particle temperature and to effectively killthe HIV virus.

The time necessary to inductively heat the minute particles near, withinor attached to the HIV virus depends substantially upon the frequencyand the power production of the alternating electromagnetic field andultimately the strength of the field produced. Similarly, for theviral-infected cells to produce alteration in cell function or celldeath, the time of treatment may vary. It should be noted, however, thatit is only necessary to raise the temperature of the virus orviral-infected cell to the minimum required to cause viral death. Thus,the concentration of particles in the vehicles and choice of variableswith respect to the type and details of electromagnetic treatment arenot critical. It is only necessary that the appropriate temperature beattained.

In a further embodiment of the invention, treatment may be accomplishedby inductive heating of magnetic particles endogenous to the HIV virusor viral-infected cells. As noted above, many types of viruses orviral-infected cells possess magnetic field-sensitive, e.g.,metal-containing compounds. Some of these are altered when the virus isin the cell and hence are more susceptible to treatment per thisinvention with an alternating electromagnetic field. Among theseparticles are storage proteins and metabolically essential porphyrinssuch as hematoporphyrins, chlorophylis, cyto-chromes or nuclear proteinmaterials. This aspect can also be based on the binding of Fe and metalions to nuclear protein which is well known. Iron-sulphur storageproteins, cytochromes or ferritin can also form the basis for thisaspect since they often differ in virus or viral-infected cells ascompared to normal host cells.

Recently it has been demonstrated that ferritin will bind to theglycoprotein envelope of the AIDS virus (HTLV virus) (Hausmann, Journ.of Virological Methods, 1987, May 16 (1-2), pgs. 125-37). This is abasis for a particularly preferred aspect of this invention. Thus,ferritin particles or ferritin attached to a magnetic particle may beused in this invention, both for diagnostic purposes as described hereinas well as for treatment with an alternating electromagnetic field.

The preferential uptake of particles by HIV virus-containing tissue isunderstandable, for example, in view of the alterations occurring inendothelial cells and the defective cells just as with endothelialgroups in AIDS lesions (Schenk et al, Archives of Oto-Rhino-Laryngology,1985, 242(3), pg. 305-13). The increased incidence of intramitochondrialiron in HIV virus-containing cells has also been demonstrated. (Sidhu G.S., et al, Human Pathology, 1985, Apr. 16 (4), pg. 377-86). Thisalteration in mitochondrial iron provides one means of utilizingendogenous ferromagnetic and/or paramagnetic centers to kill viruses orcells or to affect intracellular behavior and function invirus-containing cells. Thus, in general, uptake of virus of a cell willfurther alter and enhance its ability to selectively absorb particles.

The differences in structure between HIV virus or infected cell magneticsensitive material or metal-containing compounds and those of normalhost cells imply differences in magnetic characteristics, such asmagnetic susceptibility, between the pathogen's compounds or those ofinfected cells and those of the normal host cells. Magneticsusceptibility is known to be temperature dependent and may be routinelymeasured by magnetometer devices. By measuring the magneticsusceptibilities or other magnetic properties of particles at certaintemperatures, it is possible to calibrate the magnetometer equipment sothat measurement of magnetic susceptibility, and thus the differencesmentioned above, indicate the exact temperature of the particle inquestion. This capability may be used to selectively monitor and/or mapthe internal temperature of the invading HIV virus (as described by R.T. Gordon in U.S. Pat. Nos. 4,106,488 and 4,735,796 for the treatment ofcancer cells) by focusing on the magnetic susceptibility of amagnetically sensitive entity, e.g., a particle unique to the pathogen.Of course, as described, a precise rise in temperature sufficient tokill the pathogenic HIV virus and/or infected cells without harming thehost cells can also be effected.

The benefits of this approach to the patient are many. For example,there is little discomfort to the patient, no anesthetic is required,and the procedure is comparatively risk-free for the patient.

A further embodiment of the invention is the surface sterilization ofobjects using ferromagnetic, paramagnetic or diamagnetic particles. Inthis application of the invention, unsterile objects contaminated withHIV virus may be immersed in a solution containing said particles, whichmay be in the form of any of the compounds or elements mentioned in theprevious embodiments. The concentration of the particles in solutionwould not be critical. The contaminating viruses would, over a period oftime, take up the particles or attach the particles. A high-frequencyalternating magnetic field could then be applied to the objects, raisingthe internal temperature of the contaminating HIV organism by inductiveheating and eventually killing them. Because of the nature of thisapplication of the invention, precise control of the temperature risebelow a certain level would not be necessary, since no host cells areinvolved. This method provides a unique way of ridding objects, such assurgical instruments, of potentially dangerous viruses such asAIDS-causing viruses.

In addition, the particles of the invention may be used to diagnose,evaluate, monitor and/or locate (map) the viral infection in the hostorganism, e.g., by forming a temperature contour map. Since theparticles bind to the virus or the viral-infected cell and the particlesare also magnetically responsive, by using magnetic mapping techniques,e.g., using a magnetometer, e.g., a SQUID magnetometer, the presence andlocation of the virus can be determined. Similarly, magnetic resonanceimaging (MRI) techniques as well as electron paramagnetic resonance(EPR) and electron spin resonance (ESR) methods can be used. See, e.g.,Shaba et al., Computerized Radiol. Vol. II, No. 2, 69-73 (1987);Radiography, Jan/Feb 1986, Vol. 52, No. 60, 10.

It has been discovered also that increased oxygen delivery to enhancethe temperature rise and the energy delivery to the HIV virus as well asto the virus-infected cells can also be utilized. See the Gordon U.S.Pat. No. 4,569,836 which uses such a technique for cancer cells and doesnot imply any correlation to viruses. Nevertheless, it has beendiscovered that the methods of this patent are fully applicable herein.A hyperbaric chamber has been useful in this regard when used prior to,during or after the treatment or diagnosis of this invention. It hasbeen discovered also that chemical modification of the metabolic rate,e.g., through the use of glucose, substrate or oxidative or reductiveagents is useful to help alter the virus and virus-infected cells perthis invention. These techniques can be used to enhance virus or celldestruction. Such agents also include mitochondrial active agents whichselectively affect oxidative phosphorylation and energy activity in theviral-infected cells. A combination of such agents, such as oxygen andan agent which affects oxidative phosphorylation or other metabolicstep, or two other chemical agents per this invention, can be used toaffect the HIV virus and/or the viral-infected cells, in accordance withthis invention.

In a further advantageous aspect, the presence of the HIV virus in thecell alters its structure and the way in which the viral-infected cellbehaves and functions. Included is an alteration in the charge on thecell membrane which affects the way in which the cell takes up particleswhich enhances the effect of this invention. The virus' presence alsoenhances some receptor systems of the viral-infected cell. Similarly,there is an effect on mitochondrial structure and function, as well ason nuclear protein and other subcellular components. These effects areutilized to advantage by this invention by using the combination ofparticles and an external field to produce the desired effects and/orchemical modifications discussed herein, e.g., of the internal energylevel and/or the oxidative state of virus-infected cells, thus to alterthe virus-infected cells' function or to destroy the virus and/or theinfected cells.

This invention also relates to a variety of other advantageous methodsfor treating and/or diagnosing viruses, viral diseases and/orvirus-containing cells. For example, in accordance with this invention,viruses or viral-infected cells, particularly HIV-type viruses, can betreated by altering the oxygen delivery to the virus or viral-containingcells. In a preferred mode, the oxygen delivery is enhanced bysubjecting the cells or the host containing the cells to hyperbaric airor oxygen. This procedure, as mentioned above, is especially useful inconjunction with the heat-raising feature of this invention since theoxygen treatment enhances metabolism thereby further increasingtemperature. With regard to oxygenation of the HIV virus, see Foster etal, New York State Journal of Medicine, May 1987, 280. As with otherfeatures of this invention, this particular aspect is applicable toviruses of all sorts, i.e., both RNA and DNA viruses, and especiallyHIV-type viruses and its variants or other AIDS-causing viruses.

More generally in this regard, this invention includes methods oftreating HIV viruses or cells infected thereby by altering therespective metabolic rates by any physical or chemical means. The theoryof this treatment is essentially the same as that for the oxygenationmentioned above, i.e., increased metabolism enhances the heat-killingstep of this invention, e.g., by increasing temperature and/or producingfree radicals (analogous to singlet oxygen production above), whichcontribute to the effectiveness of weakening or destroying the virus orviral infected cell. For example, treatment may be effected by utilizingchemical or physical means to affect oxidative phosphorylation in themitochondria of vital-infected cells or by controlling glucose deliveredto such cells or to the virus per se. The former effect may be achievedfor example by monensin which is an ionophase. This agent acts to alterthe ion concentration in the mitochondria by affecting mitochondrialmembrane permeability. The latter effect can be achieved for example byusing glucose carrying agents, such as 2-deoxyglucose. The cell'smetabolic activity can also be affected by an agent such as H₂ O₂ whichwhen delivered to the cell reacts with other chemical agents alreadypresent to produce singlet oxygen.

Again, these techniques for altering the metabolic rates of viruses orviral-containing cells are especially useful in conjunction with theunderlying method of this invention involving the heat-killing ofviruses or cells infected thereby. For example, in all of these methods,one useful chemical means of altering metabolic rate is byadministration of conventional drugs which in any of a variety of waysaffect metabolic rates. For example, AZT could be coadministered inconjunction with this invention.

In another aspect of this feature, a combination of chemical agents canbe administered. Each of these can itself be active or the agents whenseparate could be inactive but when made simultaneously bioavailable,they can act together to have an effect on metabolic rate as describedabove.

In yet another aspect of this invention, the treatments of thisinvention will in fact effect a killing or other inactivation of theviruses or virus-containing cells but will not completely inactivate allof the cells. However, the mere decrease in the number of cells willenhance the ability of the body or the cells per se to immunologicallyinactivate the remaining virus. This is analogous to the situation withcancer where decreasing the tumor load helps the body develop acapability to destroy the remaining cells.

Through the use of heat per this invention to destroy the infected cellsand/or virus, immunological stimulation is definitely enhanced. Onemechanism for this enhancement is the release of immunologically activeproteins from the destroyed infected cells and/or virus. This helps inimmunological recognition of the infected cells and virus by uncoveringimmunologically important antigens.

This invention also produces other important and beneficial biophysicalalterations including, for example, intracellular production ofinterferon and interleukins based on the response of cells to thetreatment of this invention. Membrane, nuclear and cytoplasmicalterations which result therefrom are consistent with the production ofthese substances. In addition, production of these substances is anatural reaction to a foreign body type reaction. Thus, the biophysicalalterations produced by this process create a very strong foreign bodytype reaction, especially where there is a foreign particle in the cellor virus which is then activated by an external electromagnetic field toincrease the reaction even further.

In yet another feature of this invention, the various treatments and/ordiagnoses of this invention can be carried out, not only inside a body,e.g., that of a mammal, including humans, but also outside such a body.This can be effected by administering the particles to the body and thenremoving a suitable body fluid, e.g., blood, or tissue from the body andperforming a treatment or diagnosis per this invention outside the body,thereafter replacing the bodily fluid or tissue. Alternatively, thefluid or tissue can be removed from the body and then the particles canbe administered, followed by a diagnostic or treatment step of thisinvention. This aspect of the invention can be carried out fullyconventionally by utilizing readily available equipment, tubing, pumps,etc. In another alternative, the magnetic properties of the particlescan be used in conjunction with an external magnetic field to simplyremove the virus and/or viral-containing cells from the body sample.

It is also possible to affect viruses or viral-containing cells, e.g.,alter their functions or inactivate them, by applying a physical orchemical treatment which affects the dipole moment of chemical entitiesin said viruses or cells including subcellular structures. Chemicalentities having such dipole moments include substances such as ferritin,Fe-containing enzymes of the oxidative phosphorylation mechanism andother metal-containing enzymes in the cell. In addition, nucleic acidsare often associated with metals and possess a dipole moment. Thisinvention takes advantage of these entities by utilizing them to enhanceor create the effects described above. For example, through theapplication of a constant magnetic field (e.g., of 100 gauss to 80kgauss), the various dipoles in the infected virus or cell will bealigned. In addition, the dipole strengths are enhanced. Therefore, withthe resultant alignment and increased dipole moment, the interaction ofthe electromagnetic field per this invention with these dipole momentsis greatly enhanced. These dipole moments can also be affected bychemical means which are contemplated as full equivalents, e.g., theposition of protein adjuncts or other substances will affect the overalldipole of the structure. Some of these structures are smallcombi-combinations of molecules (e.g., oxidative phosphorylationenzymes) and some are large combinations (e.g., ferritin). The dipolemoment can thus be affected chemically or physically and this willassist in the interaction with the alternating electromagnetic field andwith the subsequent heating and inactivation of the infected cell and/orvirus.

Suitable static field intensities are 300-100 oersteds. The field can beapplied either before or during the treatment with the alternatingelectromagnetic field and can be used whenever one wishes to augment theeffect via the dipoles already present. Both electric and magneticfields of equivalent strengths are applicable. The advantages of thisconstant field adjunct aspect apply to all uses of this invention, e.g.,therapeutic, diagnostic, etc.

In another aspect of this invention, it has been discovered thatalterations in dielectric properties, e.g., conductivity and/orfrequency-dependent dispersion curves as a function of temperature canalso be used to measure cell temperatures and thereby map virusesanalogously to the mapping by measurement of temperature per the aboveor of magnetic properties such as magnetic susceptibility per the Gordonpatents. That is, measurements, typically along three orthogonal axes,using fully conventional equipment for measuring the dielectricproperties, conductivity and/or frequency-dependent dispersion curves,will provide the desired map using fully conventional procedures, e.g.,computer analyses.

These effects reflect the interaction of electric dipoles in tissue withthe external electric field. They are temperature-related since theorientation of the dipoles affects the temperature and/or is affected bythe temperature, e.g., the higher the temperature, the less theorientation of the dipoles. Administration of particles will affectthese properties. Analysis of these parameters, e.g., the frequencydependent dispersion curves, will reflect the changes in temperature inthe infected cells and/or viruses and/or their metabolic rates.Therefore, these measurements can also be used to monitor thetemperature and/or metabolism in the infected tissues and cells.Alterations in dielectric properties such as conductivity and frequencydispersion curves will be correlated with temperature and the metabolismof the infected cells and/or virus. As an example, the frequencydispersion curves map the response of the tissue with regard toconductivity over a given frequency range. This produces a curve ofconductivity vs. frequency. This curve will change as the temperaturechanges. By routine methods of standardization, these changes can bereadily correlated with temperature or metabolic rate changes bycomparison with a standard calibration curve constructed from a seriesof such measurements, for example. This aspect of the invention willprovide valuable information, e.g., in diagnosing viral diseases or intiming the administration according to the heating method of thisinvention. Such information will also provide valuable data regardingthe distribution of viruses in a cell population.

In yet another aspect of this invention, the particles which areadministered, instead of or in addition to being inductively heatable,will also be susceptible to acoustic waves, especially ultrasound. Theywill accordingly enhance the effect of ultrasound on the virus orviral-containing cells. This will beneficially affect intracellularand/or extracellular events. For example, the interaction of acousticwaves with tissue is related to the density and properties of the tissueinvolved, e.g., molecular spacing, inhomogeneities, etc. Through theaddition of particles, these properties can be altered. As an example,particles can increase the density of infected cells and/or viruses andhelp to increase the absorption of acoustic energy. In general,particles with a higher mass will increase the density to a greaterdegree, such as ferritin or heavy mass particles. Ultrasound will inducevibration effects on the particles and will thus serve to facilitate andto stimulate intracellular events.

The vibrational effect on the particles induced by ultrasound, and whichmay even be induced by the oscillating electromagnetic field, will serveto enhance the alteration of intracellular events as described above,including the stimulation of immunological factors. An alternatingelectromagnetic field between 1 Hz and 500 MHz can also be used toaffect said particles and make them more or less responsive to anexciting alternating electromagnetic field produced by magnetostrictiveinduced vibrations applied to said viral containing cells and viruses.Thus, the particles can be selected to include compositions thatspecifically affect intracellular and extracellular events in said viralcontaining tissue and viruses or said particles can be treated to makethem more or less responsive to an exciting alternating electromagneticfield produced by magnetostrictive induced vibrations. Furthermore, analternating electromagnetic field can be applied to said particles toproduce acoustic changes in them and affect the cellular and subcellularstructures of said viral containing tissues and/or viruses. Thesevibrational effects will also facilitate the absorption of the particlesby the cells by easing its entry through the cell membrane. Thecapability of ultrasound to affect particles is well-documented byrecent extensive work in which ultrasound is used to breakup gallstonesand kidney stones. Furthermore, the application of ultrasound in thisinvention will also have utility in the various diagnostic techniquesmentioned above to monitor the processes involved.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent.

The entire text of all applications, patents and publications, citedabove are hereby incorporated by reference.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A method of diagnosing HIV viral disease or anHIV viral infection in a cell of or from a living hostcomprising:introducing into the host of said virus or cells, particleslocalizable near, in or on the virus or vital-infected cells, suchparticles being capable of responding to a magnetic field and being of asize less than 1 micron, whereby the particles are selectively locatednear, into or on the virus and/or viral-infected cells, wherein saidparticles are magnetic metal transferrin particles, and measuring amagnetic property of the particles.
 2. A method of claim 1 wherein saidmeasuring provides a map which identifies the location and/or theconcentration of the particles.
 3. A method according to claim 2 whereina SQUID magnetometer is used for the magnetic mapping.
 4. A methodaccording to claim 2 wherein nuclear magnetic resonance is used to mapthe particles.
 5. A method according to claim 2 wherein EPR or ESR isused to map the particles.
 6. A method according to claim 1 wherein saidvirus is an AIDS-causing virus.
 7. A method according to claim 1 whereinsaid particles comprise a metal which is cobalt, zinc, chromium, nickel,platinum, manganese, a rare earth metal, dysprosium, erbium, gallium,holmium, samarium, terbium, thulium, ytterbium, yttrium or iron.
 8. Amethod of claim 1 wherein said magnetic particle is ferromagnetic,paramagnetic or diamagnetic.
 9. A method of claim 1 wherein the magneticparticle transferrin is bound to dextran.
 10. A method of claim 1wherein the particle is Fe₃ O₄ -dextran-transferrin.